Feed supplement

ABSTRACT

The present invention relates to a feed supplement or a food supplement which comprises a resin acid based composition comprising over 10% (w/w) resin acids for use in in the prevention of intestinal disorders. The invention further relates to a use of the feed supplement or the food supplement and a feed composition or a food composition comprising the feed supplement or the food supplement, respectively.

FIELD OF THE INVENTION

The invention relates to a method for preventing intestinal disorders.

BACKGROUND OF THE INVENTION

Imbalances in microbial populations and growth of harmful bacteria inthe digestive tract of animals can cause significant losses in animalgrowth and production. These imbalances manifest themselves asintestinal disorders such as diarrhea. While microbial infections ofanimals have been prevented by the use of e.g. antibiotics and otheragents that prevent the growth of microorganisms, stricter regulationson their use are expected. Ruminant animals can utilize fiber-rich rawmaterials which have little or no nutritional value for monogastricslike the human. However, the feed conversion efficiency of ruminants isrelatively low and their methane production represents a remarkableshare of the world's greenhouse gas emissions. With the increasingdemand of food there is a need to improve the feed conversion efficiencyof ruminants and to lower their methane production. Generally, there isan increasing demand for ingredients for use in animal feeding that canmodulate the microbial population in the animal digestive tract butwhich are readily available, well tolerated and environmentallyfriendly.

Fractional distillation of crude tall oil (CTO), obtained as aby-product of the Kraft process of wood pulp manufacture, producesdepitched tall oil which typically comprises over 10% resin acids andless than 90% fatty acids. Further refinement of depitched tall oilproduces tall oil fatty acid (TOFA), Distilled Tall Oil (DTO) and TallOil Rosin (TOR) which are available in a variety of compositionsdiffering in the fatty acids and resin acids content. Because TOFA is aninexpensive source of fatty acids, it has previously been used in animalnutrition as an energy source. For instance, GB 955316 discloses the useof alkali metal salts of tall oil fatty acids to improve weight gain andnitrogen retention in ruminant animals.

Toxins are poisonous substances produced within living cells ororganisms. Toxins such as mycotoxins are a chemically variable group ofsecondary metabolites of fungi, which can be found in grains and otherfeedstuffs even in the absence of any visible fungal growth. Hightemperature and air humidity during the storage increase the likelihoodof fungal growth, but mycotoxin contamination can also occur already inthe field. Visible appearance or smell of grains or silage does notindicate the presence or absence of mycotoxin contamination. Effects oftoxins such as mycotoxins to farm animals are very variable, and rangefrom increased mortality to decreased fertility and performance.Mycotoxins may also disturb the immune system of animals and make themmore susceptible to diseases.

Due to the chemical variability of mycotoxins, analysis of all feedlotsfor even the most common mycotoxins would be too expensive. Thereforemycotoxin adsorbents are often used to give extra insurance againstmycotoxin contamination in feeds. Mycotoxin adsorbents are substancesthat are itself not digested or absorbed by the animal. They are assumedto bind toxins during the passage through the alimentary canal. Thus,instead of being absorbed by the animals, the toxins get eventuallyvoided via feces.

Toxin binders can also adsorb other types of toxins, like bacterialtoxins or secondary metabolites of plants from the digestive tract.Activated carbon (charcoal) is an efficient toxin binder. It is arecommended general toxin binder in various poisonings. However,charcoal also binds vitamins and minerals, which makes it unsuitable forcontinuous use in feeds.

PURPOSE OF THE INVENTION

The purpose of the invention is to provide a new type of feed supplementor food supplement comprising resin acid based composition for use inthe prevention of intestinal disorders.

SUMMARY

The method according to the present invention is characterized by whatis presented in claim 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 The turbidity change during 8 hours of Cl. perfringens growth asa response to a Tall Oil Rosin (TOR) and Distilled Tall Oil (DTO).

FIG. 2 Gas production during 8 hours by Cl. perfringens growth as aresponse to Tall Oil Rosin (TOR) and Distilled Tall Oil (DTO).

FIG. 3A Relative gelatin degrading activity present in jejunal fromcontrol birds and birds fed the resin acid based composition-containingdiet.

FIG. 3B Relative collagen type I degrading activity present in jejunalfrom control birds and birds fed the resin acid basedcomposition-containing diet.

FIG. 3C Relative collagen type IV degrading activity present in jejunalfrom control birds and birds fed the resin acid basedcomposition-containing diet.

FIG. 3D Relative gelatin degrading activity present 789 in ileal fromcontrol birds and birds fed the resin acid based composition-containingdiet.

FIG. 3E Relative collagen type I degrading activity present in ilealfrom control birds and birds fed the resin acid basedcomposition-containing diet.

FIG. 3F Relative collagen type IV degrading activity present in ilealfrom control birds and birds fed the resin acid basedcomposition-containing diet.

FIG. 4 Effect of resin acid based composition-supplementation on ilealgelatinolytic enzymes.

DETAILED DESCRIPTION

The present invention is based on the realization that a feed supplementwhich comprises resin acid based composition can be used in theprevention of growth of harmful bacteria in the animal digestive tract,in the modulation of microbial population of the animal digestive tract,in the prevention of intestinal disorders, in enhancing rumenfermentation, lowering rumen methane production and/or in bindingtoxins.

Resin acids are present in coniferous trees, and there are three mainspecies of resin acid products, namely Tall Oil Rosin (TOR), Wood Rosinand GUM Rosin. TOR is the resin acid fraction separated by vacuumdistillation from Crude Tall Oil (CTO) which is produced by thepreparation of pulp. CTO is obtained via acidulation of Crude Tall OilSoap or Crude Sulphate Soap (TOS). TOS is separated from cooking liquidin pulp mill often called black liqueur during pulping process. WoodRosin is the fraction separated by steam distillation or other meansfrom dead trees, tree stumps, branches etc. and GUM Rosin is the resinfraction that has been steam distilled or separated by other means fromresin harvested often called tapping from a living tree.

GUM resin is widely produced in China, Indonesia and Brazil. Wood rosinmainly comes from the USA. TOR is produced in the USA and Scandinaviaand to a lesser extent in Central Europe, New Zealand and Russia.Substances containing resin acid and obtained by vacuum distillationfrom crude tall oil include Distilled Tall Oil (DTO), Tall Oil FattyAcid (TOFA) and Tall Oil Pitch (TOP). DTO contains 10-40% resin acids.CTO typically contains 15-70% resin acids, and the lowest resin acidcontents are generally provided by the cooking of mixed wood pulp.

The term “Tall Oil Rosin” or “TOR” should be understood as referring toa composition obtained by distillation of crude tall oil and furtherrefinement of distilled tall oil. TOR typically comprises 60-99% (w/w)resin acids.

The term “Wood Rosin” should be understood as referring to a compositionobtained by distillation or other means from dead trees, tree stumps,branches etc. Wood Rosin typically comprises 50-99% (w/w) resin acids.

The term “GUM Rosin” should be understood as referring to a compositionobtained by distillation or separated by other means from resinharvested from a living tree. GUM Rosin typically comprises 50-99% (w/w)resin acids.

The term “Distilled Tall Oil” or “DTO” should be understood as referringto a composition obtained by distillation of crude tall oil and furtherrefinement of distilled tall oil. DTO typically comprises 10-60% (w/w)resin acids.

The resin acid based composition TOR, Wood Rosin, GUM Rosin, CTO, TOSand DTO can also be produced by mixing one or more resin acidcompositions and one or more fatty acid compositions in form of oils orfats. Produced resin acid derivatives are for example esters, ethers oralkali metal salts.

Resin acids are known to have antimicrobial, including antibacterial,properties.

The feed supplement or the food supplement of the present inventioncomprises a resin acid based composition which comprises over 10% (w/w)of resin acids.

In one embodiment of the present invention, the feed supplement consistsof a resin acid based composition which comprises over 10% (w/w) ofresin acids. In one embodiment of the present invention, the foodsupplement consists of a resin acid based composition which comprisesover 10% (w/w) of resin acids.

In one embodiment of the present invention, the feed supplementcomprises or consists of a resin acid based composition which comprisesover 12% (w/w) resin acids. In one embodiment of the present invention,the food supplement comprises or consists of a resin acid basedcomposition which comprises over 12% (w/w) resin acids.

The feed supplement or the food supplement is effective for preventionof intestinal disorders, wherein the intestinal disorder is clinical orsub-clinical gastroenteritis, by preventing the collagen breakingactivity of matrix metalloproteinases and/or collagen breakdown in theintestinal tissues of an animal or a human, respectively.

In this context, the term “feed supplement” should be understood asreferring to a composition that may be added to a feed or used as suchin the feeding of animals. The term “feed additive” should beunderstood, unless otherwise stated, as meaning feed supplement.

In this context, the term “food supplement” should be understood asreferring to a composition that may be added to a food or used as suchin the feeding of humans.

In this context, the term “feed supplement comprising a resin acid basedcomposition” or “food supplement comprising a resin acid basedcomposition” should be understood as referring to a feed supplement or afood supplement comprising or consisting of the resin acid basedcomposition.

In this context, the term “resin acids” should be understood asreferring to a complex mixture of various acidic compounds derived fromwood, specially pine wood. They can also be modified resin acids such asdimers and decarboxylated resin acids. The exact composition of theresin acids present in the resin acid based composition varies e.g.according to the species of the trees the composition is obtained fromand the processing conditions under which it is manufactured. Resinacids typically include compounds such as abietic acid, dehydroabieticacid, levopimaric acid, neoabietic acid, pimaric acid and isopimaricacid, only to mention a few.

In the context of the feed additive, the resin acid based compositionmay be any composition described in this specification.

In one embodiment of the present invention the resin acid basedcomposition of the feed supplement comprises at least one of followingresin acids abietic acid, dehydoabietic acid, palustric acid, neoabieticacid, pimaric acid and isopimaric acid and/or derivatives thereof. Inone embodiment of the present invention the resin acid based compositionof the food supplement comprises at least one of following resin acidsabietic acid, dehydoabietic acid, palustric acid, neoabietic acid,pimaric acid and isopimaric acid and/or derivatives thereof. Thederivatives are obtained by modifying the resin acid chemically,biologically or other ways. In one embodiment of the present inventionthe resin acid based composition comprises at least one of followingresin acids abietic acid, dehydoabietic acid, palustric acid, neoabieticacid, pimaric acid and isopimaric acid. In one embodiment of the presentinvention the resin acid based composition comprises at least onechemically modified resin acid of abietic acid, dehydoabietic acid,palustric acid, neoabietic acid, pimaric acid and isopimaric acid. Theresin acid based composition may also be a mixture of unmodified andmodified resin acids.

In one embodiment of the present invention the resin acid basedcomposition of the feed supplement comprises at least three of followingresin acids abietic acid, dehydoabietic acid, palustric acid, neoabieticacid, pimaric acid and isopimaric acid and/or derivatives thereof. Inone embodiment of the present invention the resin acid based compositionof the food supplement comprises at least three of following resin acidsabietic acid, dehydoabietic acid, palustric acid, neoabietic acid,pimaric acid and isopimaric acid and/or derivatives thereof.

In one embodiment of the present invention the resin acid basedcomposition is Tall Oil Rosin (TOR).

In one embodiment of the present invention the resin acid basedcomposition and/or TOR comprises over 60% (w/w) resin acids. In oneembodiment of the present invention the resin acid based compositionand/or TOR comprises over 85% (w/w) resin acids.

The TOR can comprise 32-44.5% abietic acid, 18-25% dehydoabietic acid,0-3% dihydoabietic acid, 3.0-11.5% isopimaric acid, 0-1.5%8,5-isopimaric acid, 0-2.5% levopimaric acid, 3.3-4% neobietic acid,7.5-10% palustric acid, 3-4.5% pimaric acid and 0-4.0% sandaropimaricacid. TOR may comprise <0.1% dimers and 0-7% other components.

In one embodiment of the present invention the resin acid basedcomposition is Wood Rosin.

In one embodiment of the present invention the resin acid basedcomposition and/or Wood Rosin comprises over 10 and up to 99% (w/w)resin acids. In one embodiment of the present invention the resin acidbased composition and/or Wood Rosin comprises 50-99% (w/w) resin acids.

The Wood Rosin can comprise 45-51% abietic acid, 7.9-8.5% dehydoabieticacid, 0-1% dihydoabietic acid, 11-15.5% isopimaric acid, 0-4.2%8,5-isopimaric acid, 0-0.2% levopimaric acid, 4.7-7% neobietic acid,8.2-10% palustric acid, 3-7.1% pimaric acid and 0-2.0% sandaropimaricacid. Wood Rosin may comprise 0-4.2% dimers and 0-1% other components.

In one embodiment of the present invention the resin acid basedcomposition is GUM Rosin.

In one embodiment of the present invention the resin acid basedcomposition and/or GUM Rosin comprises over 10 and up to 99% (w/w) resinacids. In one embodiment of the present invention the resin acid basedcomposition and/or GUM Rosin comprises 50-99% (w/w) resin acids.

The GUM Rosin can comprise 15-45% abietic acid, 3-15% dehydoabieticacid, 0-0.6% dihydoabietic acid, 3.6-28% isopimaric acid, 0-0.3%8,5-isopimaric acid, 0-1.8% levopimaric acid, 10-19% neobietic acid,5-25% palustric acid, 2-7.4% pimaric acid and 0-1.5% sandaropimaricacid. GUM Rosin may comprise 0-1.0% dimers and 0-3.5% other components.

In one embodiment of the present invention the resin acid basedcomposition is Distilled Tall Oil (DTO). In one embodiment of thepresent invention the resin acid based composition is a distillationfraction of Tall Oil. In one embodiment of the present invention theresin acid based composition is a mixture of DTO and a distillationfraction of Tall Oil.

The Distillation fraction of Tall Oil is any resin acids containingfraction of CTO available during CTO refining.

In one embodiment of the present invention the resin acid basedcomposition and/or DTO comprises over 10 and up to 60% (w/w) resinacids. In one embodiment of the present invention the resin acid basedcomposition and/or DTO comprises over 10 and up to 40% (w/w) resinacids.

In one embodiment of the present invention the resin acid basedcomposition is separated from black liqueur during pulping process orTOS or CTO.

The resin acids of the resin acid based composition are insoluble inwater. The resin acids of the resin acid based composition may beunmodified or modified.

In one embodiment of the present invention the resin acids of the resinacid based composition and the feed supplement are unmodified. In oneembodiment of the present invention the resin acids of the resin acidbased composition and the food supplement are unmodified. The term“unmodified” should be understood as referring to the resin acid basedcomposition comprising over 10% (w/w) resin acids that is not modified,i.e. treated chemically, or biologically. The feed supplement or thefood supplement comprising or consisting of the resin acid basedcomposition may be used as such.

In one embodiment of the present invention the resin acids of the resinacid based composition are chemically, biologically or other waysmodified resin acid compositions. The chemical and/or biologicalmodification of resin acids of the resin acid based composition improvesthe solubility of its components and resin acids in the digestive tractof an animal. The resin acid based composition may be chemicallymodified e.g. partially or totally hydrogenated, disportinated,isomerized, oxidized, polymerized, etherified, saponified and/oresterified with suitable compounds, for example, fatty alcohols, glycol,glycerol or glyceridic fatty acid compounds such as monodi- and tri- andpolyglycerides or sugar or polyol based esters. They may be also used asa reactant in Diels-Alder reaction.

In one embodiment of the present invention, the feed supplementcomprises a resin acid based composition which is modified bysaponification. In one embodiment of the present invention, the foodsupplement comprises a resin acid based composition which is modified bysaponification.

Various processes for the saponification of the resin acid basedcomposition using e.g. NaOH or CaOH are known to a person skilled in theart. In one embodiment of the present invention, the resin acid basedcomposition for use according to the present invention is modified byetherification.

In one embodiment of the present invention the resin acid basedcomposition of the feed supplement comprises 1-90 (w/w) fatty acidsand/or their derivatives. In one embodiment of the present invention theresin acid based composition of the food supplement comprises 1-90 (w/w)fatty acids and/or their derivatives. The fatty acids may be in form ofoils or fats or in other forms like free fatty acids or esters, ethersor alkali metal salts or fatty alcohols.

In one embodiment of the present invention, the resin acid basedcomposition includes unsaponifiables which have not an acid group, forexample, lipophilic neutral substances and esters from wood. In oneembodiment of the present invention, the resin acid based compositionincludes less than 15% unsaponifiables. The amount of unsaponifiables istypically in DTO products less than 5% and in TOR, Wood and GUM Rosinless than 6%.

In one embodiment of the present invention, the feed supplementcomprises resin acid based composition which is dried. In one embodimentof the present invention, the food supplement comprises resin acid basedcomposition which is dried. The resin acid based composition can bedried by spray drying, drum drying or by any other known suitable dryingmethod.

In one embodiment of the present invention, the feed supplementcomprises different active ingredients. In one embodiment of the presentinvention, the food supplement comprises different active ingredients.

The feed supplement may be added in the feed in a concentration of0.0001-10 kg/ton of dry weight of the total amount of the feed. The feedsupplement comprising the resin acid based composition according to theinvention may be added to the feed, the food or feed supplement as such,or it may in general be further processed as desired.

The food supplement may be added in the food in an amount of 0.05-10g/day. The food supplement comprising the resin acid based compositionaccording to the invention may be added to the food or food supplementas such, or it may in general be further processed as desired.

The feed supplement or the food supplement comprising resin acid basedcomposition according to the present invention can be modified into aform which is functional and effective in feeds or foods, respectively.Carriers such as oil, fatty acids can be added to the composition forimproving the functionality. Further emulgators such as glycerols,lecithin etc. can be added to the resin acid based composition forimproving the solubility.

In one embodiment the feed supplement comprising the resin acid basedcomposition according to the present invention may comprise chemicallymodified resin acid derivatives. In one embodiment the food supplementcomprising the resin acid based composition according to the presentinvention may comprise chemically modified resin acid derivatives. Theresin acid derivatives could also comprise rosin based acid anhydrides,dimers, amines, maleimides, alkenyls, epoxy compositions and/or mixturesthereof or with other suitable chemically modified resin acids known toperson skilled in the art.

In one embodiment of the present invention, the feed supplementcomprises resin acid based composition which is absorbed into a carriermaterial suitable for the feed composition such as sugarbeet pulp.

In one embodiment of the present invention, the feed supplementcomprises resin acid based composition which is mixed with a liquidcarrier material suitable for the feed composition such as vegetableoils or fatty acids. In one embodiment of the present invention, thefood supplement comprises resin acid based composition which is mixedwith a liquid carrier material suitable for the food composition such asvegetable oils or fatty acids.

Further, the feed supplement comprising the resin acid based compositionaccording to the invention may be added to the feed, or it may beadministered to an animal separately (i.e. not as a part of any feedcomposition).

Further, the food supplement comprising the resin acid based compositionaccording to the invention may be added to the food, or it may beadministered to a human separately (i.e. not as a part of any foodcomposition).

In this context, the term “feed composition” or “feed” should beunderstood as referring to the total feed composition of an animal dietor to a part thereof, including e.g. supplemental feed, premixes andother feed compositions. The feed may comprise different activeingredients.

In this context, the term “food composition” or “food” should beunderstood as referring to the total food composition of a human diet orto a part thereof, including e.g. supplemental food, and other foodcompositions. The food may comprise different active ingredients.

The present invention also relates to a feed composition comprising thefeed supplement according to the invention.

The present invention also relates to a food composition comprising thefood supplement according to the invention.

In one embodiment of the present invention, the feed compositioncomprises the feed supplement in an amount of 0.00001-1.0% (w/w of thedry weight of the total amount of the feed.

In one embodiment of the present invention, the food compositioncomprises the food supplement in an amount of 0.00001-1.0% (w/w of thedry weight of the total amount of the food.

The present invention also relates to a use of the feed supplementaccording the present invention in a feed composition.

The present invention also relates to a use of the food supplementaccording the present invention in a food composition.

The invention also discloses a method of preventing the growth ofharmful bacteria in the animal digestive tract, comprising the step ofadministering to an animal the feed supplement comprising the resin acidbased composition according to the invention.

The invention also relates to a method of preventing intestinaldisorders, wherein the intestinal disorder is clinical or sub-clinicalgastroenteris, by preventing the collagen breaking activity of matrixmetalloproteinases and/or collagen breakdown in the intestinal tissuesof an animal or a human in need thereof, the method comprisingadministering to the animal or to the human a feed supplement or a foodsupplement, respectively comprising a resin acid based compositioncomprising over 10% (w/w) resin acids.

In the context of this method of preventing intestinal disorders, thefeed supplement or the food supplement comprising or consisting of theresin acid based composition may be any composition described in thisspecification.

In this context, the term “harmful bacteria” should be understood asreferring to any bacteria that is capable of affecting the digestivetract or health of an animal or of a human in an adverse manner,including competition for nutrients with the host animal or the human.In this context, the term “microbial population” should be understood asreferring to the microorganisms that inhabit the digestive tract,including the Bacteria and Archaea domains and microscopic members ofthe Eukaryote domain and also intestinal parasites. The microbialpopulation will vary for different animal species or humans depending one.g. the health of an animal or a human and on environmental factors.

In this context, the term “intestinal disorder” should be understood asreferring to various disorders of the digestive tract in an animal or ina human, including e.g. diarrhea and other intestinal health problems.The term “clinical or sub-clinical gastroenteritis” should be understoodas referring to an inflammation of the gastrointestinal tract—thestomach and small and large intestine. Gastroenteritis can be caused bybacteria, parasites, viruses, mycotoxins, fungus and stress.

Clinical or sub-clinical gastroenteris can be prevented by preventingthe collagen breaking activity of matrix metalloproteinases and/orcollagen breakdown in the intestinal tissues of an animal or of a human.Animal or human gut is constantly exposed to harmful molecules andmicroorganisms which endanger the integrity of the intestinal wall. Thefeed supplement or the food supplement comprising resin acid basedcomposition reduces both duodenal inflammatory T-cell infiltration andsmall intestinal matrix metalloproteinase (MMP) activity towardscollagen type I and type IV. Reduced breakdown of collagen type I and IVindicates a protective effect of resin acids on intestinal barrierintegrity by preservation of the basal membrane and the extracellularmatrix. The feed additive according to the present invention strengthensintestinal mucosal integrity and promotes intestinal health in animalsor humans.

In this context, the term “animal” should be understood as referring toall kinds of different animals, such as monogastric animals, ruminants,fur animals, pets and aquaculture. Non-limiting examples of differentanimals, including offspring, are cows, beef cattle, pigs, poultry,sheep, goats, horses, foxes, dogs, cats and fish.

In this context, the term “toxin” should be understood as referring toany poisonous substance produced within living cells or organisms.Toxins are products of plants, animals, microorganisms, for examplebacteria, viruses, fungi, rickettsiae, protozoa, etc. In this context,the term “mycotoxin” should be understood as referring to a toxicsecondary metabolite produced by fungi, such as yeast and mould. Themost common mycotoxins in grains or silage are for example aflatoxins,zearalenone, ochratoxin A, deoxynivalenol, fumonisin and T-2 toxin. Thetoxins will vary depending on environmental factors.

In one embodiment of the present invention, the resin acid basedcomposition is administered to an animal in an effective amount.

In one embodiment of the present invention, the resin acid basedcomposition is administered to a human in an effective amount.

The feed supplement comprising the resin acid based compositioncomprising over 10% (w/w) resin acids is effective in the prevention ofgrowth of harmful bacteria in the animal digestive tract, in theprevention of intestinal disorders, in the modulation of microbialpopulation of the animal digestive tract, in enhancing rumenfermentation, lowering rumen methane production and/or in bindingtoxins. They have potential in toxin binding.

The present invention has a number of advantages. The feed supplement orthe food supplement comprising the resin acid based composition is areadily available, natural, low-cost and environmentally friendlymaterial. Further, it is non-toxic and well tolerated. The feedsupplement or the food supplement comprising the resin acid basedcomposition can be used as such. The invention is effective inmodulating the composition of the microbiota in the animal or in thehuman digestive tract to a direction that is beneficial for animal orhuman performance. Subsequently, other benefits of the invention aree.g. improved animal health and productivity, higher product quality,uniformity, nutritional value and food and product safety, lower costsper production unit and decreased environmental loads. The inventionallows the production of feed compositions and supplements at low cost.

The embodiments of the invention described hereinbefore may be used inany combination with each other. Several of the embodiments may becombined together to form a further embodiment of the invention. Aproduct, a method or a use, to which the invention is related, maycomprise at least one of the embodiments of the invention describedhereinbefore.

EXAMPLES

In the following, the present invention will be described in moredetail.

Example 1

Pathogen Inhibition Test

Clostridium perfringens is a pathogenic bacterium that causes necroticenteritis in broiler chicks and other species of poultry. Thisexperiment was conducted to study the inhibition of Cl. perfringens bythe resin acid based compositions.

Two resin acid based compositions Tall Oil Rosin (TOR) and DistilledTall Oil (DTO) obtained from Crude Tall Oil distillation were tested astheir efficiency against Clostridium perfringens growth. The TORcomposition contained 88% (w/w) resin acids and the DTO compositioncontained 27.5% (w/w) resin acids.

Test compounds TOR (free resin acids 88%)  0.03 g of 1:1 in turnip rapeoil DTO (free resin acids 27.5%) 0.015 g TOR (free resin acids 88%) 0.15 ml of 10% stock solution in ethanol DTO (free resin acids 27.5%) 0.15 ml of 10% stock solution in ethanol TOR (free resin acids 88%) 0.15 ml of 1% stock solution in ethanol DTO (free resin acids 27.5%) 0.15 ml of 1% stock solution in ethanol ethanol  0.15 ml ethanol

The efficiency of test compositions was tested in a Cl. perfringensgrowth inhibition test that measures both the turbidity of theclostridial culture medium as a result of increased number of bacterialcells in a unit volume of medium, and the cumulative gas productionduring the simulation.

The efficiency of TOR and DTO against the growth of Cl. perfringens wastested at concentrations 0.01%. The TOR with 88% resin acid was meltedat +105° C. and mixed 1:1 in turnip rape oil, in order to achieve thesame runny form as the other two oily products. This diluted product wasdosed as double amount in the simulation.

Simulation procedure: The simulation was conducted in 25-ml glassbottles containing 15 ml of sterile anaerobic TSGY-media (tryptic soybroth—yeast extract media with glucose) and the bottles were enclosedwith air-tight stoppers to ensure anaerobic conditions throughout theexperiment. At the beginning of the simulation 0.1% inoculums of theovernight grown Cl. perfringens culture was injected to TSGY-bottles.Test compounds, or sterile deionized water for the control treatment,were added in a 150 μl final volume from the respective stock solutionaccording to the treatment. The simulation bottles were randomized toavoid artificial bias between treatments. The bottles were kept at aneven 37° C. temperature and mixed 1 min before the turbidity measurementat each time point. The total simulation time was 8 h.

The optical density was measured at the time points of 0.5, 4 and 8hours. The turbidity (optical density, OD) of growth media increasesproportionally as the Cl. perfringens cell number and cell densityincreases.

The total gas production was measured at the end of the 8 h simulationas an indicator of growth efficiency, since Cl. perfringens produces gasdue to the active metabolism during exponential growth.

Results

The results are illustrated in FIGS. 1 and 2. The TOR and DTO treatmentsvery effectively inhibited the growth of Cl. perfringens, which wasdetected as the lack of turbidity change (FIG. 1) and the production ofnegligible amounts of gas (FIG. 2). TOR and DTO compositions inhibitedthe growth of Clostridium perfringens very efficiently regardless of therosin acid concentration

Example 2

Methane Inhibition Test

Two resin acid based compositions Tall Oil Rosin (TOR) and DistilledTall Oil (DTO) were tested in methane inhibitio test. The TORcomposition contained 88% (w/w) resin acids and the DTO compositioncontained 27.5% (w/w) resin acids. The TOR composition containing 88%(w/w) resin acids was mixed 1:1 with turnip rape oil.

The methane inhibition test was conducted with rumen-fistulated dairycows in order to study the potential of TOR and DTO to decrease the rateof methane production in the rumen. Rumen fluid samples were measuredfor the numbers of methanogenic bacteria, as they are themethane-producing organisms. The short chain fatty acid profiles,including the concentration of branched chain fatty acids, of thesamples were measured as they indicate whether resin based acidcompositions had effects to ruminal fermentation.

Three rumen-fistulated, lactating dairy cows were given 3.0 g of drytest compositions/head/day for 21 days, in four portions. TOR and DTOcompositions were first dried onto sugar beet pulp and then mixed intothe compound feed. Rumen samples were taken before the dietaryintervention, once a week during the test composition feeding, and aftera two-week washout period. The samples of the trial were analysed forshort chain fatty acids (SCFAs) by gas chromatography and numbers ofmethanogens, protozoa and total bacteria by qPCR.

Results

The results show that the numbers of methane producing bacteriadecreased numerically during the TOR and DTO feeding period, whileprotozoa and the total number of bacteria were not affected by theproduct. The levels of lactic, propionic, and valeric acids and totalshort chain fatty acids tended to decrease in the rumen fluid during theTOR and the DTO feeding period. The concentration and relativeproportion of branched chain fatty acids tended to decrease as aresponse to TOR and the DTO.

The experiment shows that the TOR and the DTO lowers the amount ofmethanogens and thus lowers rumen methane production. The experimentalso shows that the TOR and the DTO enhances rumen fermentation.

Example 3

This experiment was conducted to study the effect of saponified DTO with35% (w/w) resin acids with or without Sugar Beet Pulp (SBP) carrier onthe microbial microbial population and fermentation of broiler chickileum in vitro.

The saponified DTO was manufactured by adding NaOH (sodium hydroxide) toDTO, adding enough water to adjust the total dry matter (DTO) percentageof the mixture to 18-20%, heating the mixture to +90° C., keeping thetemperature at +90° C. for 120 minutes, during which time the mixturewas gently stirred at 15 min intervals.

Experiment

Ileal contents of 40-days old broiler chicks were used for thesimulation media and as inoculants in the simulation models. The trialtreatments were prepared from a batch of saponified DTO.

Preparations of DTO with 35% resin acids were produced:

1. Saponified DTO with 20% dry matter content

An aliquot of the DTO soap was heated to 90° C., mixed with finelyground SBP powder, and dried.

2. Saponified DTO

Gastrointestinal digestion of the saponified DTO: Part of the liquid DTOsoap and the carrier-absorbed DTO soap was digested by apepsin-HCl-treatment (pH 2.25) followed by a pancreatin bile-acid-NaOHtreatment (pH 6.2) in a dilution series. The digestion was made toevaluate whether the products would resist the conditions of the uppergastrointestinal tract before they enter the distal intestine withhigher microbial activity.

The simulation was conducted in a total of 160 2-ml plasticmicrocentrifuge vials, in 1.5 ml volume, with 10 hours simulation time.Samples were tested at four concentrations of the dry matter of DTO: 0%,0.005%, 0.01%, 0.01% and 1%.

All the simulation samples were analysed for short chain fatty acids andthe total number of microbes. In addition, selected samples wereanalysed for a number of microbial species or groups by quantitativereal-time PCR (qPCR). Ileal simulation samples were analysed forlactobacilli and streptococci.

Results

In the ileal simulation model, DTO soap at 0.5 kg/ton level increasedthe concentrations of acetic and propionic acids and decreased theconcentration of lactic acid. This suggests modulation of microbialmetabolism from homofermentative towards heterofermentative metabolicalroute, which can be seen as a very positive change improving the feedconversion ratio. The sugar beet pulp carrier had little effect on thefermentation

Example 4

Test A: Toxin Adsorption into Solid Phase In Vitro

The capacity of a test product to remove toxins from aqueous medium wasmeasured in this test. An efficient toxin adsorbent should be able tobind the toxin in all compartments of the digestive tract, to inhibitthe toxin from getting absorbed by the animal. To evaluate the efficacyof the binder in the acidic stomach, the test was run at pH value 2.5(50 mM glycine-HCl buffer).

The test product was a saponified DTO product which contains 20% resinacids. The saponified DTO was manufactured as in example 3. The producttested was the saponified DTO (20%) with or without silicate carrier.

The test A was conducted with two toxins Ochratoxin A (OTA) andZearalenone (ZEA), at pH-value 2.5, three test substance levels 0.2, 0.5and 1 kg/ton and four replicate samples per treatment. Control treatmentwas replicated 8 times.

Mycotoxins OTA and ZEA were available as 3H-labeled pure compounds, andradioactivity, measured by liquid scintillation counting, was used fortheir quantification in the samples.

The experiment was conducted in silanized glass vials in 1 ml volume ofbuffer. In the test system, the bound radioactive toxin becomes removedfrom the liquid phase through co-pelleting with the insoluble componentsof the potential binder. The following procedure was used: 1. The testproducts were weighed into the vials, 2. 3H-labeled and intact mycotoxinwas mixed with the buffers to get the final toxin concentration of 10μg/l, 3. 1 ml of the buffer-mycotoxin solution was added to the vials,4. The vials were sealed and kept for 2 hours at 37° C. in constant slowshaking, 5. The vials were centrifuged for 10 min at 3000×g 6. 50 μl ofthe supernatant was mixed with 150 μl of liquid scintillation cocktail(Optiphase) into wells of a 96-well microtiter plate and 7. Theradioactivity of the samples was measured with a liquid scintillationcounter for five minutes

Results

The saponified DTO was able to bind OTA from the aqueous mediumstatistically significantly, and the binding was dependent on theconcentration of the test product. The saponified DTO adsorbed 25-60% ofthe free OTA from the medium.

The saponified DTO significantly decreased the amount of free ZEA evenat the lowest dosages. The saponified DTO removed approximately 30-60%of the free toxin.

Example 5

This experiment was conducted to study the effect of dietarysupplementation of resin acid based composition comprising 98.6% resinacids on broiler intestinal health under non-challenged conditions. Theexperiment focused on the effect of resin acids on collagenolyticactivities, since host metalloproteinases involved in collagen breakdownare known to play a crucial role in maintaining intestinal mucosalstructure. The resin acid based composition used in this studyrepresents the pure resin acid fraction which contains mainly abieticacid and dehydroabietic acid as shown in Table 1.

TABLE 1 The resin acid composition Major resin acids w % abietic acid47.30 dihydroabietic acid group 1.80 dehydroabietic acid 22.60neoabietic acid 0.90 dehydrodehydroabietic acid 0.80 7,9 (11)-abieticacid 5.30 13-B-7,9 (11)-abietic acid 4.50 8,12-abietic acid 1.908,15-pimaric acid 1.40 pimaric acid 0.50 isopimaric acid 3.40sandaracopimaric acid 1.40 palustric acid 6.80

Experiment

Twenty, one-day-old Ross 308 broilers were housed in two pens (10chickens per pen) on wood shavings. Water and commercial starter feedcontaining wheat, soy, corn, rice bran, wheat gluten, soy oil, corngluten, palm oil, calcium carbonate, monocalcium phosphate, corn bran,sodium chloride, sodium bicarbonate (day 1-10) or grower feed containingwheat, soy, corn, wheat gluten, sunflower seeds, soy oil, palm oil,calcium carbonate and monocalcium phosphate (day 11-22) were provided adlibitum. The control group received the standard non-supplemented diet,whereas the birds in the treatment group were fed the same feedsupplemented with 200 mg resin acid composition/kg feed throughout thewhole trial period. Upon arrival (day 1) and on days 7, 14 and 22 allbirds were weighed. At day 22, all birds were 119 euthanized forsampling. Intestinal tissue from the different segments of the smallintestine was snap frozen in liquid nitrogen and stored at −20° C. untilprotein extraction was performed for MMP analysis. Additionally,duodenal and ileal tissue samples were collected and fixed in 4%phosphate buffered formaldehyde for histological analysis.

CD3 Immunohistochemistry

Slides for immunohistochemical staining for CD3+ cells wereautomatically deparaffinized (Shandon Varistain-Gemini) before antigenretrieval with a pressure cooker in citrate buffer (10 mM, pH 6).Endogenous peroxidase activity was blocked by treating the slides withperoxidase blocking reagent (S2023, Dako, Glostrup, Denmark) for 5minutes. The presence of T-cells (CD3-positive cell abundance) inintestinal tissue from both duodenum and ileum was evaluated usingpolyclonal primary antibodies against CD3 (A0452, Dako, 1:100 dilution,30 min at room temperature), followed by incubation with a secondarylabelled polymer-HRP anti-rabbit (Envision+ System-HRP (DAB) (K4011), 30min at room temperature). Slides were evaluated using the computer basedimage analysis program, LAS V4.1. The CD3+ area percentage in either theduodenal or ileal tissue was quantified using three representativefields of view per intestinal section.

Intestinal Tissue Lysates

Proteins were extracted from the small intestinal tissue (duodenum,jejunum and ileum) using mechanical lysis. In brief, intestinal tissues(˜30 mg) were homogenized in 400 μl TBS-1% NP-40 (50 mM Tris/HCl, pH8.0, 150 mM NaCl and 1% (v/v) NP-40, supplemented with EDTA-freeprotease inhibitor cocktail (Complete, Roche, Mannheim, Germany)) bygrinding (2×) with a combination of 2.3 mm zircon/silica and 3.2 mmstainless steel beads (BioSpec Products, Bartlesville, Okla., USA) in abead beater (1.5 min, 22.5 Hz; Tissue-Lyser) with a 30 sec intervalbetween shakings. Samples were centrifuged for 10 min at 8000 rpm andthe supernatant was transferred to a new tube. Protein concentration wasmeasured using the BCA protein assay (Thermo Fisher Scientific) andsamples were stored at −20° C. until further analysis.

EnzChek Gelatinase/Collagenase Assay

The Molecular Probes EnzChek® Gelatinase/Collagenase Assay Kit was usedto evaluate the breakdown of gelatin, collagen type I and collagen typeIV by enzymes present in the small intestinal tissues (jejunum orileum). These substrates were labeled with fluorescein and a quenchingagent. Duplicate measurements were performed in 200 μl reaction volumecontaining 20 μl of either fluorescein labelled substrate (DQ Collagen I(25 μg/ml, D12060), DQ Collagen IV (25 μg/ml, D12052), or DQ Gelatin(12.5 μg/ml, D12054)), 100 μl of the tissue lysate (500 μg/ml) and 80 μlof reaction buffer (0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl2 and 2 mMsodium azide with pH 7.6). Samples were incubated for 10 h at roomtemperature in the absence of light, after which fluorescence wasmeasured (excitation 485 nm, emission 527 nm; Fluoroskan AscentFluorometer, Thermo Fisher Scientific Inc., Waltham, USA). Backgroundfluorescence was subtracted for each sample.

Gelatin Substrate Zymography

Gelatin zymography was used to identify the gelatinolytic enzymes in theileal tissue lysates. Polyacrylamide gel (10%) containing 0.2% gelatin(2 mg/ml) as substrate was used for determination of MMPs gelatinolyticactivity. Equal concentrations of ileal tissue lysates from the resinacid based composition-supplemented birds or control birds were pooled,after which 10 μl pooled ileal tissue lysate (1 mg/ml) was mixed with 10μl 2× loading buffer (0.5M Tris-HCl pH 6.8, 20% glycerol, 4% SDS, apinch of bromophenol blue) and loaded to the gel. After standardelectrophoresis, the gel was incubated with renaturing buffer (2.5%Triton X-100, 30 minutes, room temperature) to remove SDS from the gel.This allows the enzymes in the gels to renature and autoactivate. Thegel was washed with developing buffer (150 mM NaCl, 5 mM CaCl2), 0.05%NaN3 and 50 mM Tris-HCl buffer pH 7.5) and incubated with freshdeveloping buffer under continuous shaking at 37° C. for 18 hours. Afterincubation, the gel was stained with Coomassie brilliant blue(Sigma-Aldrich). Activity of gelatin-degrading enzymes is visualized ascolorless bands on a blue background. Gel images were analyzed with aGS-800 calibrated densitometer and the Quantity One software (BioRad,Hercules, Calif., USA).

Statistical Analysis

A students t-test was used for normal distributed data. When the datawere not normally distributed, the comparisons between groups were doneby Mann-Whitney U test. Analyses were done with 95% confidence intervalsand significance was determined as P≤0.05.

Results

Body Weight

Broilers receiving a diet supplemented with resin acid based compositiondid not present significant differences in body weight as compared tocontrols. No mortality was observed during the study.

Resin Acid Based Composition Reduce Duodenal T-Cell Abundance

The amount of CD3+ T-cells was determined in both the ileal and duodenaltissue as a marker for intestinal inflammation. No changes in ilealT-cell abundance were observed, whereas the amount of CD3+ positivecells in the duodenal tissue from birds fed the resin acid basedcomposition-supplemented diet was significantly decreased as compared tothe control birds as shown in Table 2.

TABLE 2 Effect of resin acid based composition- supplementation onT-cell abundance of chickens on day 22 Diet Control Resin acids P-valueCD3 area percentage Duodenum  6.78 ± 2.12  4.78 ± 1.62 0.036 Ileum 14.54± 4.05 12.24 ± 4.88 0.27

Resin Acid Based Composition-Supplementation Decreases theCollagenolytic Activity in the Broiler Ileal Tissue

The enzymatic activity towards gelatin, collagen type I or collagen typeIV present in the small intestinal tissue was assessed as a measure forextracellular matrix degradation in the gut. Jejunal (A-C) or ileal(D-E) intestinal tissue lysates from either control birds or resin acidbased composition fed birds were incubated for 10 hours withfluorescently labelled gelatin (A and D), collagen type I (B and E) orcollagen type IV (C and F). The results are illustrated in FIGS. 3A-F.In the FIG. 3 FU is relative fluorescence unit after 10 h incubation,control birds are control and resin acid based composition fed birds areresin acids. Breakdown of the fluorescently labelled substrate resultsin an increase of fluorescence which is proportional to the substratedegrading activity of the sample. In the jejunum, significantly lowergelatinase activity was measured in the tissue from resin acid basedcomposition fed birds as compared to the control group (FIGS. 3A-C). Thebiggest effect of resin acids was observed in the ileal tissue, wheregelatin, collagen type I and collagen type IV degrading activity wassignificantly decreased by supplementation of resin acid basedcomposition to the broiler feed (FIGS. 3D-F).

To gain more information on the identity of the enzymes, pooled ilealtissue lysates were subjected to gelatin zymography. The results areillustrated in FIG. 4. The ileal tissue from the control birds (control)showed three different gelatinolytic bands, whereas in the ileal tissuefrom the resin acid based composition group (resin acids), only thehighest molecular weight enzyme was present. The two enzymatic bandsthat were exclusively detected in the ileum from control birdscorrespond to MMP7 (˜18 kDa) and its latent pro-388 enzyme forms(pre-proMMP7: ˜30 kDa and proMMP7: ˜28 kDa) (UniprotKB: F6R1W4_CHICK).FIG. 4 shows that enzyme MMP7 was missing in the ileal tissue from theresin acid based composition group.

The results show that administration of resin acid composition tobroiler feed has a major effect on the host intestinal tissue. Thedietary resin acid composition supplementation resulted in decreasedabundance of inflammatory T-cells in the duodenal tissue and reducedmatrix metalloproteinase (MMP) activity, while maintaining optimalintestinal morphology. MMPs are zinc-dependent endopeptidases that areable to degrade extracellular matrix molecules as well as othermolecules that are important within the mucosal layer, such as, amongstothers, membrane receptors, adhesion factors, signaling molecules andcytoskeleton proteins. MMPs are involved in various enteric inflammatorydiseases, such as inflammatory bowel disease and necrotic enteritis inbroilers. The most profound effect of resin acid based compositionsupplementation on MMP activity was observed in the ileum, resulting ina reduction of both collagen type I and collagen type IV degradingactivity. Both collagen subtypes are important for the structuralintegrity of the intestinal wall. Collagen type I is a major supportivecomponent of the extracellular matrix, whereas type IV collagen is anintegral component of the basement membrane supporting the epithelialcells. The main enzyme responsible for the reduced collagenolyticactivity in the resin acid based composition had a molecular weightcorresponding to both the latent and active forms of MMP7. In thehealthy intestine, various MMPs are expressed, but the production ofMMP7 is mainly linked to injured epithelium. MMP7 can disrupt epithelialbarrier integrity by degrading intercellular junction molecules such ascadherins and occludins. Furthermore, MMP7 is able to activateα-defensins by cleaving its precursor into the active form. Alphadefensins are antimicrobial peptides that are secreted by epithelialcells and granulocytes, which play a role in protection of the hostagainst microbial invasion during intestinal inflammation and can induceIL6 secretion by macrophages, thereby contributing to intestinal leakageand inflammation. As the broiler gut is continuously exposed to variouschallenges that affect intestinal barrier integrity and can triggerinflammation (e.g. coccidia, mycotoxins, bacterial toxins, amongstothers) reducing MMP7 activity through resin acid basedcomposition-supplementation to the diet supports animal performance byenhancing intestinal barrier integrity and controlling inflammation inthe avian gut.

The resin acid based composition-supplementation of broiler diet reducedduodenal inflammatory T-cell abundance and small intestinal MMPactivity.

It is obvious to a person skilled in the art that, with the advancementof technology, the basic idea of the invention may be implemented invarious ways. The invention and its embodiments are thus not limited tothe examples described above; instead they may vary within the scope ofthe claims.

The invention claimed is:
 1. A method for preventing intestinaldisorders, wherein the intestinal disorder is clinical or sub-clinicalgastroenteritis caused by Clostridium perfringens, by preventing thecollagen breaking activity of matrix metalloproteinases and/or collagenbreakdown in the intestinal tissues of an animal or a human in needthereof, the method comprising administering to the animal or to thehuman a feed supplement or a food supplement, respectively, comprising aresin acid based composition comprising over 12% (w/w) resin acids. 2.The method according to claim 1, wherein the method comprisesadministering to the animal a feed supplement comprising a resin acidbased composition comprising over 10% (w/w) resin acids.
 3. The methodaccording to claim 1, wherein the resin acid based composition comprisesat least one of following resin acids abietic acid, dehydoabietic acid,palustric acid, neoabietic acid, pimaric acid and isopimaric acid and/orderivative thereof.
 4. The method according to claim 1 wherein resinacid based composition comprises at least three of following resin acidsabietic acid, dehydoabietic acid, palustric acid, neoabietic acid,pimaric acid and isopimaric acid and/or derivative thereof.
 5. Themethod according to claim 1, wherein resin acid based compositioncomprises or consists of Tall Oil Rosin (TOR), Wood Rosin, GUM Rosinand/or Distilled Tall Oil (DTO).
 6. The method according to claim 5,wherein the resin acid based composition is Tall Oil Rosin (TOR).
 7. Themethod according to claim 5, wherein the resin acid based compositioncomprises over 60% (w/w) resin acids.
 8. The method according to claim5, wherein the resin acid based composition is Wood Rosin or GUM Rosin.9. The method according to claim 8, wherein the resin acid basedcomposition comprises over 10 and up to 99% (w/w) resin acids.
 10. Themethod according to claim 5, wherein the resin acid based composition isDistilled Tall Oil (DTO).
 11. The method according to claim 10, whereinthe resin acid based composition comprises over 10 and up 60% (w/w)resin acids.
 12. The method according to claim 1, wherein the resinacids are unmodified.
 13. The method according to claim 1, wherein theresin acid based composition is absorbed into a carrier material ormixed with a carrier.